title: A novel glutamate transporter blocker, LL-TBOA, attenuates ischaemic injury in the isolated, perfused rat heart despite low transporter levels.
abstract: OBJECTIVE
  Loss of glutamate from cardiomyocytes during ischaemia may aggravate ischaemia-reperfusion injury in open heart surgery. This may be due to reversal of excitatory amino acid transporters (EAATs). However, the expression of such transporters in cardiomyocytes is ambiguous and quantitative data are lacking. Our objective was to study whether EAATs were expressed in the rat heart and to study whether blocking of transporter operation during cardiac ischaemia could be beneficial.
  
  
  METHODS
  We used TaqMan real-time PCR and immunoisolation followed by western blotting to unequivocally identify EAAT subtypes in rat hearts. We used a novel high-affinity non-transportable competitive inhibitor, named LL-TBOA [(2S,3S)-3-(3-(6-(6-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)-ethoxy)ethoxy) acetamido)hexanamido)- hexanamido)-5-(4-(trifluoromethyl)benzamido)benzyloxy) aspartic acid], to block EAAT-mediated transport during global ischaemia and reperfusion of isolated rat hearts.
  
  
  RESULTS
  Rat hearts expressed EAAT subtypes 1 and 3, while subtypes 2 and 4 were not detected. Hearts were isolated and perfused with 1.6 µM LL-TBOA for 5 min before 30 min of induced global ischaemia and 60 min of reperfusion (n = 8). Control hearts were perfused either with the solvent dimethylsulfoxide 3.5 mM (n = 7) or with no pretreatment (n = 8). Infarct size was evaluated by triphenyl tetrazolium chloride (TTC) staining. LL-TBOA reduced infarct size from 33 ± 14 to 20 ± 5% (mean ± SD) (P = 0.015). Dimethylsulfoxide alone had no effect (35 ± 2%). Reperfusion arrhythmias were reduced by LL-TBOA (P = 0.009), but not by dimethylsulfoxide alone.
  
  
  CONCLUSIONS
  Rat hearts express EAAT1 and EAAT3, but the mRNA levels are, respectively, ∼ 25 and 200 times lower than in the brain. Addition of LL-TBOA has a beneficial effect against ischaemia-reperfusion injury.
authors.affiliation: Department of Physiology, Institute of Basic Medical Science, University of Oslo, Oslo, Norway.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Martinov
authors.forename: Yvette
authors.initials: Y
authors.lastname: Dehnes
authors.forename: Silvia
authors.initials: S
authors.lastname: Holmseth
authors.forename: Keiko
authors.initials: K
authors.lastname: Shimamoto
authors.forename: Niels Christian
authors.initials: NC
authors.lastname: Danbolt
authors.forename: Guro
authors.initials: G
authors.lastname: Valen
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000037:antagonists & inhibitors*; D000818:Animals; D001145:Arrhythmias, Cardiac / Q000188:drug therapy* / Q000517:prevention & control; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use; D018691:Excitatory Amino Acid Antagonists / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D006352:Heart Ventricles / Q000187:drug effects; D009203:Myocardial Infarction / Q000188:drug therapy; D015428:Myocardial Reperfusion Injury / Q000188:drug therapy* / Q000473:pathology; D051381:Rats
chemical_list: D027322:Amino Acid Transport System X-AG; D018691:Excitatory Amino Acid Antagonists; D001224:Aspartic Acid
keywords: Ischaemia-reperfusion; Myocardial protection; Reperfusion arrhythmias